Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms including fungi, and mycelial bacteria, in particular the actinomycetes. An appreciation for the wide variety of polyketide structures, and for their biological activities, may be gained upon review of the extensive art, for example, published International Patent Specifications WO 93/13663 and WO 95/08548; U.S. Pat. Nos. 5,098,837, 5,149,639, 4,874,748, 5,063,155; and the journal articles H. Fu et al., Biochemistry, 33, pp. 9321-9326, (1994); R. McDaniel et al., Science, 262, pp. 1546-1550, (1993); and J. Rohr, Angew. Chem. Int. Ed. Engl. 34(8), pp.881-888, (1995).
Polyketides are synthesized in nature on polyketide synthases ("PKS"). These enyzmes, which are actually complexes of multiple enzyme activities, are in some ways similar to, but in other ways different from, the synthases which catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. Two major types of PKS are known which are very different in their construction and mode of synthesis. These are commonly referred to as Type I or "modular" and Type II "aromatic."
The PKS enzyme complexes that are the subject of the present invention are members of the group designated Type I or modular PKS. In this type of PKS, a set of separate catalytic active sites (each active site is termed a "domain", and a set thereof is termed a "module") exists for each cycle of carbon chain elongation and modification. Upon inspection of the structure of a polyketide it is generally possible to determine the number and nature of the PKS modules necessary to form the polyketide, although the number of polypeptides that provide the modules may remain unknown, as may the exact nature of the starter unit.
FIG. 9 of aforementioned WO95/08548 depicts a typical genetic model for a Type I PKS, in this case for 6-deoxyerythronolide B synthase ("DEBS") involved in the production of erythromycin. Six separate modules, each catalyzing a round of condensation and modification of a 2-carbon unit, are present. The number and type of catalytic domains that are present in each module varies (see the WO 95/08548 FIG. 9) based on the needed chemistry, and the total of 6 modules is provided on 3 separate polypeptides (designated DEBS-1, DEBS-2, and DEBS-3, with 2 modules per each). Each of the DEBS polypeptides is encoded from a separate open reading frame (gene), see Caffrey et al., FEBS Letters, 304, pp. 205, 1992.
The catalytic domains of the DEBS polypeptides provide a representative example of Type I PKS design. In this particular case, modules 1 and 2 reside on DEBS-1, modules 3 and 4 on DEBS-2, and modules 5 and 6 on DEBS-3, wherein module 1 is defined as the first module to act on the growing polyketide backbone, and module 6 the last.
A minimal PKS module may be typified by module 3 which contains a ketosynthase ("KS") domain, an acyltransferase ("AT") domain, and an acyl carrier protein ("ACP") domain. These three enzyme activities are sufficient to activate the 2-carbon extender unit and attach it to the growing polyketide molecule. Additional domains that may be included in a module relate to reactions other than the actual condensation, and include a ketoreductase activity ("KR") activity, a dehydratase activity ("DH"), and an enoylreductase activity ("ER"). With respect to DEBS-1, the first module thereof also contains repeats of the AT and ACP activities because it catalyzes initial condensation, i.e. it begins with a "loading domain" represented by AT and ACP, which determine the nature of the starter unit. The "finishing" of the 6-deoxyerythronolide molecule is regulated by a thioesterase activity ("TE") in module 6. This thioesterase appears to catalyze cyclization of the macrolide ring thereby increasing the yield of the particular polyketide product.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker or "scaffold"-encoding regions. These scaffold regions encode amino acid sequences that space the enzymatic activities (domains) at the appropriate distances and in the correct order. Thus, these linker regions collectively can be considered to encode a scaffold into which the various domains (and thus modules) are placed in a particular order and spatial arrangement. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) between PKS species by various available methodologies. Thus, there is considerable flexibility in the design of new PKS systems with the result that known polyketides can be produced more effectively, and novel polyketide pharmaceuticals can also be made.
As aforementioned, an additional level of structural complexity in the resultant polyketides may be introduced by subsequent glycosylation or other post-PKS chemical or enzymatic reactions.
DNA sequences that encode the novel PKS of the present invention may be included in a variety of host cells, there resulting novel recombinant host cells for the production of polyketides. Representative examples include those mentioned in U.S. patent application Ser. No. 09/114,083, filed Jul. 10, 1998, for example with reference to plant cells, and international patent publication WO 98/27203 where the examples of bacterial and yeast cells may be mentioned, the text of each application being incorporated by reference herein as if fully set forth. Additional suitable host cells include, for example, animal cells, and particular bacteria such as E. coli, Streptomyces, and Sorangium. According to the practice of the invention, particular host cells are selected on the basis of their capacity to facilitate expression of PKS enzymes, the capacity of such cells to produce (including secrete) polyketides, and the nature of the intracellular environment which may determine which substrates, including primers, are made available for reaction.